The long-term objective of this project is to achieve a biochemical description of the mechanisms underlying protein traffic between membrane-bounded compartments of eukaryotic cells. Two distinct GTP- dependent Golgi Binding Factors, or GGBFs, were purified from tissue on the basis of their ability to cause GTPgammaS-dependent block of a cell- free assay that reconstitutes intra-Golgi transport. Both GGBFs are members of the ADP-ribosylation factor family of GTPases, but have different activities in the Golgi transport assay. The ARF family contains a large number of related proteins that may be involved in a wide range of cellular reactions. Most of our efforts will concentrate on elucidating the function of GGBFs in intra-Golgi transport and testing the possibility that each member of this family has a specialized function using both biochemical and genetic approaches. The specific aims of the project can be summarized as follows. (1) Further characterize GGBFs and related members of the ARF family using GTP-binding and cell-free transport assays in order establish if different ARFs have specialized function. (2) Identify GGBFs by microsequencing, clone their cDNAs and overexpress proteins in E. coli. (3) Raise polyclonal and monoclonal antibodies by immunization with pure protein or peptides derived from unique sequences of GGBFs. (4) Determine whether GGBFs are required to observe intra-Golgi transport and/or to produce vesicle intermediates. (5) Identify novel transport factors on the basis their effect on GGBF-activity and on the binding and hydrolysis of GTP by GGBFs (or on the basis of their association with GTP-bound GGBF). (6) Select "RNA antibodies" using SELEX as a way of circumventing current problems in obtaining specific and neutralizing antibodies to ARFs. Determine function(s) of GGBFs in vivo either by expressing high levels of inhibitory RNAs, or by reducing GGBF mRNA levels using antisense oligodeoxynucleotides. Members of the ARF family are likely to be involved in controlling assembly of components of the intracellular transport machinery. The work proposed in this application will lead to a more in-depth understanding of the function of these proteins. Furthermore, the identification and characterization of proteins with which they interact will contribute significantly towards a description of the mechanisms regulating intracellular traffic.